The increasing number of reports of direct transmission of avian influenza viruses to humans underscores the need for control strategies to prevent an influenza pandemic. Vaccination is the key strategy to prevent severe illness and death from pandemic influenza. Despite long-term experience with vaccines against human influenza viruses, researchers face several additional challenges in developing human vaccines against avian influenza viruses. In this Review, we discuss the features of avian influenza viruses, the gaps in our understanding of infections caused by these viruses in humans and of the immune response to them that distinguishes them from human influenza viruses, and the current status of vaccine development.

In late November 2014 higher than normal death losses in a meat turkey and chicken broiler breeder farm in the Fraser Valley of British Columbia initiated a diagnostic investigation that led to the discovery of a novel reassortant highly pathogenic avian influenza (HPAI) H5N2 virus. This virus, composed of 5 gene segments (PB2, PA, HA, M and NS) related to Eurasian HPAI H5N8 and the remaining gene segments (PB1, NP and NA) related to North American lineage waterfowl viruses, represents the first HPAI outbreak in North American poultry due to a virus with Eurasian lineage genes. Since its first appearance in Korea in January 2014, HPAI H5N8 spread to Western Europe in November 2014. These European outbreaks happened to temporally coincide with migratory waterfowl movements. The fact that the British Columbia outbreaks also occurred at a time associated with increased migratory waterfowl activity along with reports by the USA of a wholly Eurasian H5N8 virus detected in wild birds in Washington State, strongly suggest that migratory waterfowl were responsible for bringing Eurasian H5N8 to North America where it subsequently reassorted with indigenous viruses.

Avian influenza A H7 subtype viruses pose a significant threat to human health because of their ability to transmit directly from domestic poultry to humans and to cause disease and, sometimes, death. Although it is important to develop vaccines against viruses of this subtype, very limited information is available on the immune response and pathogenesis of H7 viruses in animal models such as mice and ferrets. Ten H7 viruses were selected for possible vaccine development on the basis of their phylogenetic relationships and geographical locations. The virulence of the 10 viruses for mice and the immunogenicity of the viruses in mice and ferrets were evaluated to study the extent of antigenic relatedness and the level of cross-reactivity of antibodies. Most of the viruses showed similar patterns of cross-reactivity with mouse and ferret antisera. The Eurasian viruses elicited broadly cross-reactive antibodies that neutralized viruses from both Eurasian and North American lineages, but the converse was not true. A subset of the viruses was also evaluated for the ability to replicate and cause disease in BALB/c mice following intranasal administration. H7 subtype viruses were able to infect mice without adaptation and manifested different levels of lethality and kinetics of replication. On the basis of phylogenetic data, induction of broadly cross-neutralizing antibodies in mouse and ferret antisera, and their ability to replicate in mice, we have selected A/Netherlands/219/03 (subtype H7N7) and A/chicken/BC/CN-7/04 (subtype H7N3) viruses for vaccine development. The mouse model can be used for the preclinical evaluation of these vaccines against H7 subtype viruses.

The appearance of human infections caused by avian influenza A H7 subtype viruses underscores their pandemic potential and the need to develop vaccines to protect humans from viruses of this subtype. A live attenuated H7N3 virus vaccine was generated by reverse genetics using the HA and NA genes of a low pathogenicity A/chicken/BC/CN-6/04 (H7N3) virus and the six internal protein genes of the cold-adapted A/Ann Arbor/6/60 ca (H2N2) virus. The reassortant H7N3 BC 04 ca vaccine virus was temperature sensitive and showed attenuation in mice and ferrets. Intranasal immunization with one dose of the vaccine protected mice and ferrets when challenged with homologous and heterologous H7 viruses. The reassortant H7N3 BC 04 ca vaccine virus showed comparable levels of attenuation, immunogenicity and efficacy in mice and ferret models. The safety, immunogenicity, and efficacy of this vaccine in mice and ferrets support the evaluation of this vaccine in clinical trials.

Piscine reovirus (PRV) was common among wild and farmed salmonids in British Columbia, western Canada, from 1987 to 2013. Salmonid tissues tested for PRV by real-time rRT-PCR included sections from archived paraffin blocks from 1974 to 2008 (n = 363) and fresh-frozen hearts from 2013 (n = 916). The earliest PRV-positive sample was from a wild-source steelhead trout, Oncorhynchus mykiss (Walbaum), from 1977. By histopathology (n = 404), no fish had lesions diagnostic for heart and skeletal muscle inflammation (HSMI). In some groups, lymphohistiocytic endocarditis affected a greater proportion of fish with PRV than fish without PRV, but the range of Ct values among affected fish was within the range of Ct values among unaffected fish. Also, fish with the lowest PRV Ct values (18.4-21.7) lacked endocarditis or any other consistent lesion. From 1987 to 1994, the proportion of PRV positives was not significantly different between farmed Atlantic salmon, Salmo salar L. (44% of 48), and wild-source salmonids (31% of 45). In 2013, the proportion of PRV positives was not significantly different between wild coho salmon, Oncorhynchus kisutch (Walbaum), sampled from British Columbia (5.0% of 60) or the reference region, Alaska, USA (10% of 58).

In Manitoba, Canada, several swine herds were infected by pandemic (H1N1) 2009 virus in the summer of 2009. Results of several investigations concluded that outbreaks of infection with this virus are similar in duration to outbreaks of infections with swine influenza viruses A (H1N1) and A (H3N2).

Development of live attenuated influenza vaccines (LAIV) against avian strains with pandemic potential is an important public-health strategy. Either 1 or 2 107-TCID50 doses of H9N2 LAIV A/chicken/Hong Kong/G9/97 were administered intranasally to 50 adults in isolation; 41 participants were H9N2 seronegative, 24 of whom received 2 doses. The vaccine was well tolerated; vaccine shedding was minimal. After 2 doses, 92% of H9-seronegative participants had ≥4-fold increases in hemagglutination-inhibition antibody, and 79% had ≥4-fold increases in neutralizing antibody; 100% had responses detected by at least 1 assay. Although replication of the H9N2 LAIV was restricted, 2 doses were immunogenic in H9N2-seronegative adults.

Rabies is a fatal zoonotic disease that requires fast, accurate diagnosis to prevent disease in an exposed individual. The current gold standard for post-mortem diagnosis of human and animal rabies is the direct fluorescent antibody (DFA) test. While the DFA test has proven sensitive and reliable, it requires high quality antibody conjugates, a skilled technician, a fluorescence microscope and diagnostic specimen of sufficient quality. The LN34 pan-lyssavirus real-time RT-PCR assay represents a strong candidate for rabies post-mortem diagnostics due to its ability to detect RNA across the diverse Lyssavirus genus, its high sensitivity, its potential for use with deteriorated tissues, and its simple, easy to implement design. Here, we present data from a multi-site evaluation of the LN34 assay in 14 laboratories. A total of 2,978 samples (1,049 DFA positive) from Africa, the Americas, Asia, Europe, and the Middle East were tested. The LN34 assay exhibited low variability in repeatability and reproducibility studies and was capable of detecting viral RNA in fresh, frozen, archived, deteriorated and formalin-fixed brain tissue. The LN34 assay displayed high diagnostic specificity (99.68%) and sensitivity (99.90%) when compared to the DFA test, and no DFA positive samples were negative by the LN34 assay. The LN34 assay produced definitive findings for 80 samples that were inconclusive or untestable by DFA; 29 were positive. Five samples were inconclusive by the LN34 assay, and only one sample was inconclusive by both tests. Furthermore, use of the LN34 assay led to the identification of one false negative and 11 false positive DFA results. Together, these results demonstrate the reliability and robustness of the LN34 assay and support a role for the LN34 assay in improving rabies diagnostics and surveillance.